Cell Science at a Glance 4873 APC at a glance to the development of cancer and to this region of APC include the that interactions between APC and regulatory subunit of phosphatase 2a Inke Näthke cytoskeletal proteins are also important. (PP2A), APC-stimulated guanine Cancer Research UK, Senior Research Fellow, Cell nucleotide exchange factor (ASEF) for & Developmental Biology, University of Dundee, Rho family proteins, and KAP3, which Dow Street, Dundee, DD1 5EH, UK The APC protein is a linker protein for kinesin motor (e-mail: [email protected]) Cancer-associated mutations in APC proteins (Bienz, 2002; Dikovskaya et al.,

Journal of Cell Science 117, 4873-4875 Published by usually lead to the expression of N- 2001). Whether all these interactions can The Company of Biologists 2004 terminal fragments. This region of the occur simultaneously and how they are doi:10.1242/jcs.01313 APC protein contains heptad repeats regulated is not known. that are predicted to form coiled-coil The gene encoding the adenomatous domains and might be involved in The middle of the APC molecule polyposis coli protein (APC) is mutated oligomerisation (Joslyn et al., 1993). contains domains important for in most colon cancers. The major role of The N-terminal region of APC also interactions with proteins in the WNT APC is thought to be as a scaffold for contains two nuclear export signals signalling pathway (Rubinfeld et al., a protein complex that regulates the (NES) that are required for shuttling of 1996). This region includes three 15- phosphorylation and thus degradation APC between the nucleus and cytoplasm residue repeats that constitutively bind to of β-catenin in the WNT signalling and may directly interact with exportin β-catenin and seven 20-residue repeats pathway (Huelsken and Behrens, 2002). Crm-1 (Henderson and Fagotto, 2002). that also bind to β-catenin but are However, there is increasing evidence regulated by phosphorylation (Rubinfeld that dysregulation of β-catenin is not the Embedded in the heptad repeats of APC et al., 1996). In addition, it contains three only effect of mutations in APC relevant are armadillo repeats. Proteins that bind stretches that are involved in binding to

Subconfluent cell

Microtubules WNT

Kap3/KIF MT EB1 binding

Armadillo 15 aa 20 aa Oligomerisation repeats (x7) repeats repeats SAMP repeats Basic domain

Coiled coil GSK3β B56 of PPase β-Catenin β-catenin Axin/conductin

PDZ domain binding ASEF NES WNT NLS DNA Cortex/F-actin

Confluent cells jcs.biologists.org

β Apical APC -catenin Kap3 E-APC GSK3β Kif3 F-Actin Axin EB1 WNT MT Dsh AJ Dlg Asef Middle

+

+ + + + + Basal Inke Näthke

 Journal of Cell Science 2004 (117, pp. 4873-4875)

(See poster insert) 4874 Journal of Cell Science 117 (21) axin (Bienz, 2002). The N-terminus and destruction by the proteasome. In the ends efficiently. This suggests that a middle region of APC are the most presence of a WNT signal, the protein combination of direct and indirect highly conserved regions in the Dishevelled (Dsh) inactivates GSK3β. interactions of APC with microtubules sequences of APC proteins from This results in a decrease in the amount determine its peculiar distribution (Barth different species (http://www.rzpd.de/ of β-catenin targeted for degradation, et al., 2002; Zumbrunn et al., 2001). To cgi-bin/cards/carddisp?APC). thus increasing the amount available complicate matters further, the same to activate TCF/LEF transcription region of APC that binds to KAP3 also The C-terminal region of APC contains factors. stimulates ASEF (Kawasaki et al., motifs that mediate interactions with a 2000), which suggests that APC can also number of structural proteins: a basic Truncation mutations of APC that render stimulate actin polymerisation locally. stretch enriched in positively charged it unable to bind and recruit β-catenin This may explain why APC can be residues is similar to the microtubule- lead to an accumulation of β-catenin, detected in association with actin-rich binding site of the tau protein and which correlates with changes in structures (Rosin-Arbesfeld et al., 2001). represents the major microtubule- transcriptional activation by TCF/LEF The ability to bind to other actin- binding site of APC; the terminal 170 transcriptions factors and the expression associated proteins, including PDZ- residues can bind to EB1, a small of a variety of genes that can change the domain-containing proteins such as Dlg, microtubule-end-binding protein; the C- proliferation and differentiation state of may further contribute to the interaction. terminal 15 residues constitute a binding cells, including those encoding MYC, site for PDZ domains (Dikovskaya et al., cyclin D, ephrins and caspases (Chen et The intricate interplay of different 2001). al., 2003; Fodde, 2002; van de Wetering protein interactions involving APC et al., 2002). However, there are suggests that the distribution of APC The C-terminal third of APC proteins is additional pathways that contribute to between specific intracellular networks the least well conserved between species the regulation of β-catenin and are is determined by a variety of signals that and the second, smaller APC proteins – independent of APC and other proteins depend on the cellular environment. For E-APC and APC-2/L – do not contain a involved in the WNT pathway. These instance, in polarised epithelial cells, basic domain that is readily identifiable include the p53-inducible Siah-1 protein APC appears to be weakly associated as a microtubule-binding site, an EB1- (Liu et al., 2001) and a retinoid X with F-actin structures but is mostly binding site or the C-terminal motif that receptor (Xiao et al., 2003). The concentrated at the basal region of the mediates binding to PDZ domains complicated relationships between these plasma membrane, where the plus ends (Dikovskaya et al., 2001). The role of pathways make it difficult to establish of the microtubules that form large these alternative APC proteins in human exactly how the deregulation of β- parallel arrays in fully polarised cancer remains elusive. catenin that result from mutations in epithelial cells terminate (Mogensen, APC contributes to the initiation of 2002). In fact, APC may be involved in colon cancer. This in turn supports the establishing such large microtubule Functions of APC idea that other functions of APC are arrays since in APC+/– mice the number The best-characterised function of APC involved in its role in cancer. of microtubules in such arrays is is as a scaffolding protein in a multi- significantly reduced (Mogensen, 2002). protein complex whose activity is The importance of interactions between modulated by WNT signalling (Fodde, APC and the cytoskeleton are reflected A lack of APC leads to defects in the 2002). This complex regulates the by the distribution of the endogenous structure of mitotic spindles that can phosphorylation of β-catenin and thus APC protein: in sub-confluent, migrating only be rescued by APC containing the controls the amount of β-catenin epithelial cells, APC predominantly direct microtubule-binding site; this is available for transcriptional activation localises to the peripheral ends of a consistent with a role for APC in via TCF/LEF transcriptions factors. subset of microtubules and accumulates establishing microtubule arrays Other proteins that are part of this in distinct clusters near the plasma (Dikovskaya et al., 2004). Furthermore, complex include GSK3β, β-catenin, membrane that coincide with areas of in early mitosis, APC localises to the axin along with several kinases and active cell migration, although the F- ends of microtubules that are embedded phosphatases. A number of reviews actin content at these sites is low (Näthke at kinetochores, but also decorates describe the details of this pathway et al., 1996). In polarised cells, the spindle microtubules and centrosomes (Bienz, 2002; Fodde, 2002; Huelsken highest concentration of APC is detected (Dikovskaya et al., 2004; Fodde et and Behrens, 2002), making an in-depth at the basal surface where the plus ends al., 2001; Kaplan et al., 2001; Louie discussion here not necessary. Briefly, of microtubules terminate (Mogensen et et al., 2004). Loss of APC leads in the absence of extracellular WNT al., 2002). to chromosome mis-segregation, signals, GSK3β in the APC–β- suggesting that mutations in APC catenin–axin complex is active, Although the ability of APC to interact contribute to cancer by causing phosphorylating all three of these with KAP3 is at least partially aneuploidy (Dikovskaya et al., 2004; proteins, which increases their responsible for its distribution, Fodde et al., 2001; Kaplan et al., 2001). interaction. This phosphorylation of β- fragments of APC that can bind to KAP3 Interestingly, N-terminal fragments that catenin creates a recognition site for but lack the direct microtubule-binding result from mutations in APC are not ubiquitin ligases and leads to its site do not localise to microtubule plus only unable to support the formation of Cell Science at a Glance 4875 normal mitotic spindles (because they do I apologise to those whose work was not Akiyama, T. (2000). Asef, a link between the not bind to and stabilise microtubules) mentioned here due to severely limited space. I am tumor suppressor APC and G-protein signaling. particularly grateful to Ewan E. Morrison for Science 289, 1194-1197. but also appear to exert a dominant effect donating the image of APC in subconfluent cells, Liu, J., Stevens, J., Rote, C. A., Yost, H. J., Hu, on mitotic spindles (Green and Kaplan, Ian Newton and Karin Kroboth for preparing Y., Neufeld, K. L., White, R. L. and Matsunami, 2003). confluent cells for microscopy and Dina N. (2001). Siah-1 mediates a novel beta-catenin Dikovskaya for critical comments on the degradation pathway linking p53 to the manuscript. adenomatous polyposis coli protein. Mol. Cell 7, APC is also found in the nucleus and 927-936. contains several nuclear import and Louie, R. K., Bahmanyar, S., Siemers, K. A., export signals (Henderson and Fagotto, References Votin, V., Chang, P., Stearns, T., Nelson, W. J. 2002). One function suggested for and Barth, A. I. (2004). Adenomatous polyposis Barth, A. I., Siemers, K. A. and Nelson, W. J. coli and EB1 localize in close proximity of the nuclear APC is the support of nuclear (2002). Dissecting interactions between EB1, mother centriole and EB1 is a functional shuttling of β-catenin. However, microtubules and APC in cortical clusters at the component of centrosomes. J. Cell Sci. 117, 1117- nuclear-cytoplasmic shuttling of β- plasma membrane. J. Cell Sci. 115, 1583-1590. 1128. catenin can proceed independently of Bienz, M. (2002). The subcellular destinations of Mogensen, M. M., Tucker, J. B., Mackie, J. B., APC proteins. Nat. Rev. Mol. Cell. Biol. 3, 328- Prescott, A. R. and Nathke, I. S. (2002). The APC (Henderson and Fagotto, 2002). 338. adenomatous polyposis coli protein Other potential functions for APC in the Chen, T., Yang, I., Irby, R., Shain, K. H., Wang, unambiguously localizes to microtubule plus ends nucleus include a contribution to H. G., Quackenbush, J., Coppola, D., Cheng, J. and is involved in establishing parallel arrays of transcriptional regulation via its ability Q. and Yeatman, T. J. (2003). Regulation of microtubule bundles in highly polarized epithelial caspase expression and apoptosis by adenomatous cells. J. Cell Biol. 157, 1041-1048. to bind DNA directly (Deka et al., 1999). polyposis coli. Cancer Res. 63, 4368-4374. Näthke, I. S., Adams, C. L., Polakis, P., Sellin, Deka, J., Herter, P., Sprenger-Haussels, M., J. H. and Nelson, W. J. (1996). The adenomatous The relative amount of APC in the Koosch, S., Franz, D., Muller, K. M., Kuhnen, polyposis coli tumor suppressor protein localizes nucleus may be related to cell cycle stage C., Hoffmann, I. and Muller, O. (1999). The to plasma membrane sites involved in active cell APC protein binds to A/T rich DNA sequences. migration. J. Cell Biol. 134, 165-179. since cells at low confluency exhibit Oncogene 18, 5654-5661. Roberts, G. T., Davies, M. L. and Wakeman, J. more staining for APC in the nucleus Dikovskaya, D., Zumbrunn, J., Penman, G. A. A. (2003). Interaction between Ku80 protein and a compared with densely growing cells and Näthke, I. S. (2001). The Adenomatous widely used antibody to adenomatous polyposis (Fagman et al., 2003; Zhang et al., Polyposis Coli protein, in the limelight out at the coli. Br. J. Cancer 88, 202-205. 2001); however, nuclear staining edge. Trends Cell Biol. 11, 378-384. Rosin-Arbesfeld, R., Ihrke, G. and Bienz, M. Dikovskaya, D., Newton, I. P. and Näthke, I. S. (2001). Actin-dependent membrane association of observed using many APC antibodies (2004). The adenomatous polyposis coli protein is the APC tumour suppressor in polarized has to be interpreted carefully because required for the formation of robust spindles mammalian epithelial cells. EMBO J. 20, 5929- antibody cross-reactivity can contribute formed in CSF Xenopus extracts. Mol. Biol. Cell 5939. significantly to staining of APC in the 15, 2978-2991. Rubinfeld, B., Albert, I., Porfiri, E., Fiol, C., Fagman, H., Larsson, F., Arvidsson, Y., Munemitsu, S. and Polakis, P. (1996). Binding of nucleus. Indeed, at least one Meuller, J., Nordling, M., Martinsson, T., GSK3beta to the APC-beta-catenin complex and commercially available APC antibody Helmbrecht, K., Brabant, G. and Nilsson, M. regulation of complex assembly. Science 272, reacts strongly with the DNA-binding (2003). Nuclear accumulation of full-length and 1023-1026. protein Ku80 and only very weakly with truncated adenomatous polyposis coli protein in van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., van der APC (Mogensen et al., 2002; Roberts et tumor cells depends on proliferation. Oncogene 22, 6013-6022. Horn, K., Batlle, E., Coudreuse, D., Haramis, A. al., 2003). Fodde, R. (2002). The APC gene in colorectal P. et al. (2002). The beta-catenin/TCF-4 complex cancer. Eur. J. Cancer 38, 867-871. imposes a crypt progenitor phenotype on colorectal In summary, by interacting with a Fodde, R., Kuipers, J., Rosenberg, C., Smits, R., cancer cells. Cell 111, 241-250. Xiao, J. H., Ghosn, C., Hinchman, C., Forbes, complex set of cellular proteins and Kielman, M., Gaspar, C., van Es, J. H., Breukel, C., Wiegant, J., Giles, R. H. et al. (2001). C., Wang, J., Snider, N., Cordrey, A., Zhao, Y. pathways, APC contributes to Mutations in the APC tumour suppressor gene and Chandraratna, R. A. (2003). Adenomatous differentiation, cell migration, cause chromosomal instability. Nat. Cell Biol. 3, polyposis coli (APC)-independent regulation of proliferation and adhesion. The toxic 433-438. beta-catenin degradation via a retinoid X receptor- mediated pathway. J. Biol. Chem. 278, 29954- environment of the gut lumen means that Green, R. A. and Kaplan, K. B. (2003). Chromosome instability in colorectal tumor cells is 29962. all cells other than stem cells have a associated with defects in microtubule plus-end Zhang, F., White, R. L. and Neufeld, K. L. short life span in this tissue. Active attachments caused by a dominant mutation in (2001). Cell density and phosphorylation control migration accompanies cell APC. J. Cell Biol. 163, 949-961. the subcellular localization of adenomatous Henderson, B. R. and Fagotto, F. (2002). The ins polyposis coli protein. Mol. Cell. Biol. 21, 8143- differentiation to ensure that epithelial 8156. cells in the gut are usually exfoliated and outs of APC and beta-catenin nuclear transport. EMBO Rep. 3, 834-839. Zumbrunn, J., Inoshita, K., Hyman, A. A. and within 3-5 days. As a consequence, Huelsken, J. and Behrens, J. (2002). The Wnt Näthke, I. S. (2001). Binding of the adenomatous normal gut maintenance requires that signalling pathway. J. Cell Sci. 115, 3977-3978. polyposis coli protein to microtubules increases Joslyn, G., Richardson, D. S., White, R. and microtubule stability and is regulated by GSK3b cell-cell and cell-substrate adhesion phosphorylation. Curr. Biol. 11, 44-49. along with migration, proliferation and Alber, T. (1993). Dimer formation by an N- terminal coiled coil in the APC protein. Proc. Natl. differentiation are balanced and Acad. Sci. USA 90, 11109-11113. maintained at all times. Mutations in Kaplan, K. B., Burds, A., Swedlow, J. R., Bekir, Cell Science at a Glance on the Web APC are likely to affect all of these S. S., Sorger, P. K. and Näthke, I. S. (2001). A Electronic copies of the poster insert are processes, which may explain why novel role for the APC tumour suppressor in available in the online version of this article mutations in this single gene are chromosome segregation. Nat. Cell Biol. 3, 429- at jcs.biologists.org. The JPEG images can 432. be downloaded for printing or used as sufficient to initiate the development of Kawasaki, Y., Senda, T., Ishidate, T., Koyama, slides. cancer. R., Morishita, T., Iwayama, Y., Higuchi, O. and